Nic Tapon: Projects

Metazoan organ development requires a strict control of cell proliferation, growth and death. Studies in Drosophila melanogaster have identified the Hippo (Hpo) pathway as one of the major signalling pathways required for tissue size control.

The Hpo pathway controls the final tissue and organ size by both inhibiting cell proliferation and promoting apoptosis. At the core of the pathway lies a kinase cascade comprised of the Ste20-related kinase Hpo and the Dbf2-related kinase Warts (Wts).

Hpo and Wts, together with their respective scaffold proteins Salvador (Sav) and Mob as Tumour Suppressor (Mats), phosphorylate and inhibit the transcriptional co-activator Yorkie (Yki). Recent work in vertebrates indicates that the growth control function of Hpo signalling is conserved and is relevant to mammalian tumour formation.

The key open question in Hpo signalling remains what cues control its activity, to ensure precisely the correct amount of growth inhibitory signal to specify organism size. The upstream regulation of the Hpo pathway is complex, integrating many inputs.

Firstly, through the KEM (Kibra/Expanded/Merlin) and AMOT complexes, which associate with the apical polarity protein Crumbs (Crb), the basolateral determinant Scribble and junctional components such as α-catenin, Hpo signalling is believed to respond to cell-cell contact integrity and apico-basal polarity. Indeed, the Hpo pathway is a mediator of contact inhibition of growth in cell culture and many mutations that perturb cell-cell contacts or polarity lead to de-repression of Yki target genes.

Secondly, the atypical cadherins Fat (Ft) and Dachsous (Ds) are thought to repress the core kinase cascade by inactivating the myosin Dachs and the LIM domain protein Zyxin.

Thirdly, mechanical forces, sensed through the actin cytoskeleton, have also been shown to modulate Yki/YAP activity, but the molecular mechanism remains unclear.

Finally, G-protein coupled receptors (GPCRs) have recently been shown to modulate Hpo pathway activity through at least two distinct mechanisms. Thus, the Hpo pathway is believed to respond to a variety of signals such as local tissue architecture (cell crowding, tissue mechanics) as well as diffusible signals (GPCR ligands).

Regulation of Hippo signalling by the Salt-inducible kinases

In order to shed light on Hpo pathway upstream signalling, we have performed a genome-wide RNAi screen in cell culture in collaboration with the High-Throughput Screening Facility at the LRI headed by Mike Howell, and Moritz Rossner's lab at the Max Planck Institute in Göttingen.

The screen is based on the Split-TEV (Tobacco Etch Virus) technology developed in the Rossner lab, which allows us to detect the phospho-dependent interaction between Yki and 14-3-3. Using this approach, we identified the Salt-inducible kinases (Sik) as regulators of Hpo signalling. Activated Sik2 and 3 kinases increase Yki target expression and promote tissue overgrowth through inhibitory phosphorylation of Sav at Serine 413.

Siks play a major role in inhibiting gluconeogenesis in the liver in response to high glucose levels through inhibitory phosphorylation of the transcriptional co-activator CRTC2 (CREB-regulated transcription coactivator 2)/TORC2, and activatory phosphorylation of the Histone Deacetylase HDAC4, a function which appears to be conserved in Drosophila.

The Siks are under hormonal control by Insulin receptor (InR) signalling and Glucagon (Adipokinetic Hormone (AKH) in flies). InR activates Akt, which phosphorylates and activates Sik2/3. Glucagon signals through a G-protein coupled receptor (GPCR), inducing PKA activation, which phosphorylates and inhibits Sik2/3.

Under fasting conditions in flies, low Insulin and high AKH activity combine to inhibit Sik3, thereby promoting gluconeogenesis and inducing mobilisation of Fat Body (FB - the fly liver equivalent) lipid stores to restore circulating glucose levels and energy homeostasis. Thus, we hypothesise that Sik kinases may provide a nutritional/metabolic input to Hpo signalling, ensuring that Yki drives tissue growth only under favourable conditions.

Differential proliferation rates generate patterns of mechanical tension that orient tissue growth

Orientation of cell divisions is a key mechanism of tissue morphogenesis. In the growing Drosophila wing imaginal disc epithelium, most of the cell divisions in the central wing pouch are oriented along the proximal-distal (P-D) axis by the Ds-Ft-Dachs planar polarity pathway. However, cells at the periphery of the wing pouch instead tend to orient their divisions perpendicular to the P-D axis despite strong Dachs polarisation. In collaboration with Barry Thompson ( Epithelial Biology Group), Alexander Tournier (Mathematical Modelling Group) and Andreas Hoppe (Kingston University, London), we have shown that these circumferential divisions are oriented by circumferential mechanical forces (as measured by laser ablation experiments - Figure 1) that influence cell shapes and thus orient the mitotic spindle.

Figure 1

Figure 1. Stills from a movie of a laser ablation used to visualise differential tensions experienced by cell junctions in Drosophila larval wing epithelial cells. The arrowhead in A indicates the ablated junction. The frames in A. and B. show the cell junction before and after the cut, respectively. C. is a merge of A. and B.

We propose that this circumferential pattern of force is not generated locally by polarised constriction of individual epithelial cells. Instead, these forces emerge as a global tension pattern that appears to originate from differential rates of cell proliferation within the wing pouch. Accordingly, we show that localised overgrowth is sufficient to induce neighbouring cell stretching and reorientation of cell division. Our results suggest that patterned rates of cell proliferation can influence tissue mechanics and thus determine the orientation of cell divisions and tissue shape.

Recent theoretical work has suggested that mechanical feedback control of proliferation is a likely mediator of terminal proliferation arrest during wing disc development. In particular, the pattern of compression in the centre and stretching in the periphery has been proposed to account for equilibrating the differences in pro-growth signals between the centre of the pouch (distal), where cells are exposed to high levels of the Dpp and Wg morphogens versus the periphery (proximal) where cells are exposed to lower morphogen levels. Yki (YAP in mammals) has been reported to respond to a cell's mechanical environment and might function as a growth-regulatory sensor of these physical inputs. While this is an attractive hypothesis, it was unclear how the patterns of stretch and compression observed in late discs arise in the first place.

Our data suggest that early differences in proliferation rates in the centre versus the periphery likely account for these patterns, which might feed back to increase proliferation in stretched outer cells, leading to proliferation rate equilibration. It will be interesting in the future to explore whether differential growth rates can influence the spatial pattern of Yki activation in vivo.

Nic Tapon

nic.tapon@crick.ac.uk
+44 (0)20 379 62050

  • Qualifications and history
  • 1997 PhD in Biochemistry, University College London, UK.
  • 1998 Postdoctoral Fellow, Massachusetts General Hospital Cancer Centre, USA
  • 2003 Established lab at the London Research Institute, Cancer Research UK
  • 2015 Group Leader, the Francis Crick Institute, London, UK